There is a drive in the pharmaceutical industry towards the development of bispecific therapeutics that can concurrently bind two or more distinct targets or epitopes in order to achieve novel mechanisms of action and efficacy. See, Beck et al., 2010, Nature Reviews, Immunology 10: 345-352; Carter, 2011, Experimental Cell Research 317: 1261-1269; Kontermann, 2012, mABs 4: 182-197; and Segal et al., 2001, Journal of Immunology Methods 248:1-6. In recent years, a number of bispecific formats based on either antibody or other protein domains have been designed with the goal of creating a modular molecular scaffold. See, Kontermann, 2012, mABs 4:182-197; and Klein et al., 2012, mABs 27:4(6). From this, it is clear that modular multi-domain, multi-functional monoclonal antibodies, with their intrinsic therapeutically relevant features combined with the experiences gained in the biopharmaceutical development of these molecules as therapeutics, makes this class of molecules an attractive molecular class for pharmaceutical development provided that such molecules do not substantially deviate from their native structural and functional characteristics.
Initial IgG-like bispecific antibody development centered on use of a hybrid hybridoma of two cells that produces two different antibodies of interest. See, Milstein and Cuello, 1983, Nature 305: 537-540. Co-expression of the four different antibody chains (two heavy and two light) in such a fused cell leads to the non-selective formation of up to ten different combinations of heavy and light chain pairs, from which the one correct bispecific molecule is recovered through laborious purification. Improving on this, some workers have used either natural or engineered differences in Protein A binding affinities of the two antibody heavy chains for selective isolation of the heterodimer from the homodimers. See Lindhofer et al., 1995, Journal of Immunology 155: 219-225; Igawa and Tsunoda, 2007, United States Patent Publication No. 2009/0263392 A1; Davis and Smith, 2010, “Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format”, United States Patent Publication No. 2010/00331527; and Klein et al., 2012, MAbs. 27:4(6). The bispecific antibody of interest that is obtained in any of these non-selective chain pairing expression strategies appears to be limited to a maximum of 12.5% of the total antibody yield in cases where both light-heavy and heavy-heavy chain pairing is essential or 50% if selective light-heavy chain pairing requirement is abrogated such as by using a common light chain. In either case this approach will significantly impact the cost of goods.
In order to overcome this impact and diminish the formation of unwanted Fc chain pairs, structure guided attempts to engineer mutations resulting in selective pairing of preferred heavy chains when co-expressed in a recombinant manner is desirable. Prominent among these rational design efforts is the knob-into-hole strategy, developed by Presta, Carter and coworkers, which employs steric point mutations in the CH3-CH3 interface to preferentially drive Fc heterodimerisation and prevent formation of homodimers. See, Ridgway and Presta, 1996, Protein Engineering 9: 617-621; Merchant et al. 1998, Nature Biotechnology 17: 677-681; and Atwell et al., 1997, Journal of Molecular Biology 270: 26-35. Such designs have yielded high heterodimer selectivity, but have caused about 11° C. lowering in thermal stability of the CH3 domain relative to the wild type. In contrast to this steric complementarity approach in the knob-into-hole designs, Gunasekaran and coworkers have recently employed electrostatic complementarity design strategy to achieve the selective heterodimerization goal. See, Gunasekaran et al., 2010, The Journal of Biological Chemistry 285: 19637-19646. Davis and coworkers have designed strand exchange engineered domain (SEED) CH3 which is comprised of alternating segments of human IgA and IgG CH3 sequences leading to preferentially associating heterodimers. See Davis et al., 2010, PEDS 23: 195-202. The engineered CH3 domains of both these approaches have melting temperatures of the CH3 domains of ˜68° C.
Alternately, an annealing based approach for producing bispecific antibodies by mixing two different antibodies has been pursued in other technologies. See Jackman et al., 2010, J. Biol. Chem 285: 20850-20859; and Strop et al., 2012, J. Mol. Biol. 420, 204-219. These rational engineering approaches favor heterodimer formation by destabilizing the natural homodimer interface and result in antibodies comprising less stable CH3 domains than the parent molecule. A protein with reduced stability of its native folded state is potentially prone to a number of aggregation related challenges in its handling and development. See, Wang, 2005, International Journal of Pharmaceutics 289: 1-30; and Demarest et al., 2008, Current Opinion in Drug Discovery and Development 11: 675-687. Further, the mutations in the IgG Fc region and the reduced stability of the CH3 domain could have an impact on immunogenicity and pharmacokinetic properties, which are important drug like properties that have to be validated for successful design of a modular bispecific scaffold.
Given the above background, there is a need in the art for Fc heterodimer proteins in crystalline form, cystallizable compositions comprising such Fc heterodimer proteins, and methods for identifying mutations which promote heterodimeric Fc chain pair formation. Such articles and methods are needed in order to develop polypeptide constructs that comprise antigen-binding domains that are linked to an Fc heterodimer protein comprising CH3 domains which have been modified to select for heterodimers with favorable drug-like properties such as ease of manufacturing and analytical characterization; formulation and stability of the therapeutic at the requisite drug concentrations; and pharmacokinetic properties, immunogenicity and toxicity that are similar to Fc heterodimer proteins without a modified CH3 domain. An antibody platform that takes into consideration all of these aspects concurrently would significantly empower the drug developer in the design of best-in-class bi- and multi-specific therapeutic candidates.